I.McFarlane at icrf.icnet.uk (Ian McFarlane) wrote:
>My protein of interest happens to be soluble in 10% TCA. I am going to use
>TCA as the first step in its pufn. Does anyone know if I can go on to do
>hydrophobic interaction chromatography without dialysing away the TCA.
>Obviously anion or cation exchange would require dialysis. Any
>suggestions?
I have never tried HIC from TCA solutions. However, if this does not
work, there are at least two options other than dialysis. The first would
be gel filtration, which at the same time as removing TCA can be used as
second purification step (by MW, obviously). The other option would be
ultrafiltration in a stirred cell, like the ones produced by Amicon. The
solute is pressed through a membrane by nitrogen pressure, proteins above
the cut off size of the membrane are retained. Simply concentrate your
sample to, say, 1/10 or 1/20 of its original volume, add your starting
buffer for the next step and concentrate again. This process is a lot
quicker than dialysis (normally 2-3 h total) and gives you a concentrated
sample, which is easier to work with (although this is not too relevant
with HIC).